For centuries, calf rennet has been used as a milk coagulant in the production of all varieties of cheese. In face, cheeses made with this enzyme set the standards for flavor, appearance and texture with which cheeses made from other coagulants are compared. Recently, dramatic increases in world cheese production and decreases in calf rennet supplies have stimulated the use of alternative milk coagulant enzymes.
Among the available enzymes for this purpose, the microbial rennets are favored because they can be mass produced and offer a variety of properties permitting selection of those most suitable in cheese production. Unfortunately, the increased thermal stability, especially when compared to calf rennet, of microbial rennets, such as the Mucor microbial rennets, has been considered to be a less desirable property of these enzymes.
In the cheese-making process, the whey that separates from the rennet-coagulated curd is collected as a valuable source of whey proteins. Normally, the collected whey is pasteurized, at a temperature from about 60.degree. C. to 71.degree. C., for a sufficient time to destroy any microorganisms and to thermally inactivate at least about 80 to 90% of any residual rennet coagulant. Residual levels of coagulant enzyme retaining much more than about 20% of their original milk-clotting activities after normal pasteurization have been found to undesirably hydrolyze the valuable whey proteins and restrict the use of the whey as an ingredient in various food products. The normal pasteurizing conditions, which are sufficiently mild to leave the whey proteins unaffected but sufficiently severe to thermally inactivate calf rennet, are much less effective to inactivate microbial rennet coagulants to the desired level. This problem is further complicated by the fact that more microbial rennet is partitioned into the whey than is calf rennet. As a result there has been a continuing effort to improve the thermal lability of microbial rennet.
Several microbiological approaches to obtain more heat-labile microbial coagulants have been tried. These approaches include comprehensive screens for new microorganisms and mutations of known rennet-producing microorganisms. To date, neither approach has been successful in obtaining an acceptable microbial coagulant with the desired thermal properties.
Chemical approaches to modify various properties of enzymes have also been tried but, in general, are severely limited by a concomitant loss in the enzyme's milk-clotting activity. Recent studies, however, to determine the structural and functional determinants of one Mucor microbial rennet indicate that some chemical modification of microbial rennet may not result in the total loss of milk coagulant activity. Such modifications include nitration (Biochemica et Biophysica Acta, 371, 368 [1974]), carbamylation (ibid., 271, 93 [1972] and periodate-induced deglycosylation (ibid., 328, 52 [1973]) of the glycoenzyme from Mucor microbial rennet. Of these modifications, there is some suggestion that carbamylation and periodate-induced deglycosylation may impart some increased thermal lability. Unfortunately, these chemical approaches have not been completely successful in producing a microbial rennet with the desired degree of thermal lability.
It is therefore highly desirable to produce a microbial rennet with desirable milk coagulant properties but with substantially increased thermal labile properties.